Method of producing l-histidinol by fermentation

ABSTRACT

Histidinol is accumulated in histidine-containing, but otherwise conventional aqueous culture media on which histidine-requiring mutants of Brevibacterium, Corynebacterium, or Arthrobacter are cultured, and is readily recovered from the fermentation broth.

United States Patent Kubota et al.

[451 May 9,1972

[54] METHOD OF PRODUCING L- HISTIDINOL BY FERMENTA'IION [72] Inventors:Koji Kubota, Kawaski-shi; Teruo Shiro,

Chigasaki-shi, both of Japan [73] Assignee: Ajinomoto Co., Inc., Tokyo,Japan [22] Filed: Dec. 23, 1968 21 Appl. No.: 786,361

[30] Foreign Application Priority Data Dec. 30, 1967 Japan ..42/85303 52us. Cl ..195/30 [51] Int. Cl. ..Cl2b l/OO [58] Field of Search.....l95/28, 29, 30, 42, 43, Si

Kloos et al. J. Gen. Microbiol, Vol. 39, p. 185- 194; 1965 Vol. Vogel eta], J.A.C.S.; Vol. 73; p. 1897- 8; 1951 Primary Examiner-A. LouisMonacell Assistant Examiner-Gary M. Nath An0rneyKurt Kelman ABSTRACTHistidinol is accumulated in histidine-containing, but otherwiseconventional aqueous culture media on which histidinerequiring mutantsof Brevibacterium, Corynebacterium, or Arthrobacter are cultured, and isreadily recovered from the fermentation broth.

7 Claims, No Drawings METHOD OF PRODUCING L-HISTIDINOL BY FERMENTATIONThis invention relates to the ferrnentative production of L- histidinol,and in particular, to a method of producing L- histidinol at low costfrom readily available raw materials.

L-l-listidinol has been used in medical research and as an intermediatein the production of histidine, urocanic acid, and histamine.

It has been known since 1951 (J.A.C.S. 73, 1897) that ahistidine-requiring mutant strain of Escherichia coli can produceextracellular L-histidinol. However, the amount of L- histidinolaccumulating in the culture medium is too small to apply this method onan industrial scale.

We have now found that histidine-requiring mutant strains of the generaBrevibacterium, Corynebacterium and Arthrobacter produce and accumulatesubstantial amounts of L-histidinol in economically useful amounts whencultured aerobically on otherwise conventional culture media containinghistidine.

The mutant strains are readily obtained in a conventional manner byexposing vegetative cells or spores of the parent to ultraviolet light,to X-rays, or to gamma rays, and by screening the irradiated materialfor its response to histidine. Suitable mutant strains have also beenobtained by contacting the parent strain with sodium nitrite solution ina well known manner.

Brevibacterium flavum ATCC 21349, Corynebacterium acetoacidophilum ATCC21350, and Arthrobacter citreus ATCC 21348 are representativemicroorganisms which may be employed in the present method. They may beobtained from the American Type Culture Collection in Washington, DC.

The culture media employed for producing L-histidinol by our method maybe entirely conventional. They must contain an assimilable carbonsource, an assimilable nitrogen source, growth promoting organicsubstances, and inorganic salts or ions. Suitable carbon sources includecarbohydrates, such as glucose, fructose,'maltose, sucrose, xylose,starch hydrolyzate, molasses, and organic acids such as acetic acid andcitric acid. Alcohols and hydrocarbons have also been employedsuccessfully as carbon sources.

Nitrogen may be provided by ammonium salts of inorganic acids such asammonium sulphate and ammonium chloride, or by ammonia in aqueoussolution or in the gaseous state which may be used simultaneously for pHcontrol in a known manner. Organic nitrogen-bearing compounds such asamino acids, urea or protein hydrolyzate may also provide assimilablenitrogen. I

Supplemental inorganic nutrients required include the essentialinorganic ions available from potassium phosphate, magnesium sulfate,manganese sulfate, zinc sulfate, ferrous sulfate, sodium chloride, andsodium carbonate.

Organic growth promoting substances which improve the yield and the rateof production of L-histidinol include vitamins and fatty acids, and maybe added to the culture medium in the form of substances which yield theactive agent under the conditions of fermentation, such as peptone,yeast extract, corn steep liquor, soybean protein hydrolyzate, andvarious other extracts of vegetal and animal tissues, well known inthemselves. Histidine should be present in the culture medium in apreferred concentration of 5 to 50 mg. per deciliter. At lowerconcentrations, the growth rate of the microorganisms and the rate ofL-histidinol production are very low, and at higher concentrations, thegrowth of the microorganisms normally proceeds rapidly, but the rate ofL-histidinol production is reduced.

For a good yield of L-histidinol, the fermentation should be carried outwith aeration and agitation in order to supply sufficient oxygen to thebroth. Best yields of L-histidinol cannot be obtained unless the pHvalue of the culture medium is controlled between 5 and 9. Aqueousammonia, gaseour ammonia, calcium carbonate, or alkali metal hydroxidesmay be added to the nutrient medium from time to time, as required,

to maintain the desired pH range. For best results, the temperature ofthe broth should be held between 25 and 37 C. during fermentation. Thefermentation is preferably carried out for 2 to 5 days.

The recovery of L-histidinol from the culture broth may follow knownmethods. The bacterial cells may be removed by filtration orcentrifuging, and L-histidinol may be recovered by employing anion-exchange resin in combination with treatment by active carbon and aprecipitation method.

The product of the present method has been identified as L- histidinolby the Rf value of its paper chromatogram, the color development bydiazo reaction of the paper chromatogram, and the fact thathistidine-requiring microorganisms which can also grow on a mediumcontaining L-histidinol can grow on a conventional medium to'which aneluate of the paper chromatogram was added.

The L-histidinol accumulated in the broth was determined quantitativelyby Macphersons colorimetric method (Biochemical Journal Vol. 40, page470, 1946).

The following Examples are further illustrative of the present inventionand it will be understood that the invention is not limited thereto.

EXAMPLE 1 An aqueous culture medium was prepared from sucrose 7 percent,KH PQ, 0.2 percent, MgSO.,'7l-l O 0.04 percent, ammonium sulfate 1.0percent, dry yeast 1.0 percent, and polypepton 0.2 percent, and 20 mlbatches of the solution were placed in 500 ml. shaking flasks andsterilized at 1 15 C. for 5 minutes. Separately sterilized calciumcarbonate was thereafter added in an amount of 2.5 percent to each batchin order to adjust the pH to 6.5.

The aqueous media were inoculated with Brevibacterium flavum ATCC 21349,which had been previously cultured on bouillon slants at 30 C. for 24hours. The fermentation was carried out at 28 C. for 70 hours. 2.3 g.L-histidinol (as dihydrochloride) were formed'per liter of the broth.

The microbial cells were removed from 5 liters of the broth bycentrifuging, and the L-histidinol in the solution was absorbed oncarbon powder at pH 4 and eluted with a mixture of percent ethanol and15 percent ION hydrochloric acid. The eluate was partly evaporated toremove the alcohol and the concentrate was diluted with water, adjustedto pH 1.0, and passed over a column packed with a cation exchange resin(Dowex-SO, H type). The column was washed with water, and theL-histidinol was then eluted with l.5N hydrochloric acid and 2.5Nhydrochloric acid. The eluate was partly evaporated in a vacuum and 5.6g. crude, crystalline L-histidinol dihydrochloride were precipitatedfrom the residual liquid by addition of cool alcohol.

EXAMPLE 2 An aqueous culture medium was prepared from glucose 10percent, KH PQ, 0.1 percent, MgSO -7H O 0.04 percent, ammonium sulfate4%, biotin 10 ug/dl, thiamine hydrochloride 20 ,ug./dl, Fe 2 ppm, Mn 2ppm. and histidine hydrochloride 25 mg percent. Its pH was adjusted to7.5 with aqueous ammonia. 20 Milliliter batches of the solution wereplaced in 500 ml. shaking flasks, and were sterilized by steam at 110 C.for 5 minutes. They were then inoculated with Brevibacterium flavum ATCC21349, which had been previously cultured as described in Example 1, andthe fermentation was carried out at 3 1 .5 C. for 55 hours. 5.8 g.L-histidinol (as di-hydrochloride) were found in each liter of thebroth.

EXAMPLE 3 An aqueous culture medium was prepared to contain sucrose 7percent, KH PQ, 0.15 percent, MgSO.,-7H O 0.04 percent, diammoniumhydrogen phosphate 1.5 percent, biotin ug/l, thiamine hydrochloride 500,ug/l, iron ion 2 ppm, manganese ion 2 ppm, yeast extract 0.1 percent,soy bean protein hydrolyzate 4 percent, and calcium carbonate(sterilized separately) 3 percent, and its pH was adjusted to 7.0. 20

' Milliliter batches of the solution were placed in 500 ml. shak-EXAMPLE 4 An aqueous culture medium was prepared to contain glucose 10percent, KH PO 0.1 percent, MgSO -7H O 0.04 percent, ammonium sulfate 3percent, biotin 50 p.g./l, thiamine hydrochloride 200 ug/l, iron ion 2p.p.m., manganese ion 2 p.p.m., L-histidine hydrochloride 20 mg.percent, soy bean protein hydrolyzate 1 percent, and calcium carbonate(sterilized separately) percent, and its pH was adjusted to 7.5. 20Milliliters batches of the solution were placed in 500 ml shakingflasks, and were sterilized by steam at 110 C. for 5 minutes. They werethen inoculated with Arthrobacter citreus ATCC 21348, which had beenpreviously cultured as described in Example 3. The fermentation wascarried out at 30 C. for 72 hours. 4.6 Grams per liter L-histidinol (asdihydrochloride) were found in the broth.

What we claim is:

l. A method of producing L-histidinol which comprises:

a. culturing a histidine-requiring microorganism of the group consistingof Brevibacterium flavum ATCC 21349. Corynebacterium acetoacidophilumATCC 21350, and Arthrobacter citreus ATCC 21348 under aerobic conditionsin a medium containing an assimilable carbon source, an assimilablenitrogen source, essential inorganic salts, and an effective amount ofhistidine, while maintaining the culture medium at a pH of 5 to 9, untilI..- histidinol accumulates in the medium; and

b. separating the accumulated L-histidinol from said medi- 2. A methodas set forth in claim 1, wherein said histidine is present in saidmedium in an amount of 5 to 50 milligrams per deciliter.

3. A method as set forth in claim 1, wherein the histidinerequiringstrain is Brevibacterium flavum ATCC 21349.

4. A method as set forth in claim 1, wherein the histidinerequiringstrain is Corynebacterium acetoacidophilum ATCC 21350.

5. A method as set forth in claim 1, wherein the histidinerequiringstrain is Arthrobacter citreus ATCC 21348.

6. A method as set forth in claim 1, wherein said L-histidinol isrecovered in the form of a crystalline salt thereof.

7. A method as set forth in claim 6, wherein said salt is thehydrochloride.

2. A method as set forth in claim 1, wherein said histidine is presentin said medium in an amount of 5 to 50 milligrams per deciliter.
 3. Amethod as set forth in claim 1, wherEin the histidine-requiring strainis Brevibacterium flavum ATCC
 21349. 4. A method as set forth in claim1, wherein the histidine-requiring strain is Corynebacteriumacetoacidophilum ATCC
 21350. 5. A method as set forth in claim 1,wherein the histidine-requiring strain is Arthrobacter citreus ATCC21348.
 6. A method as set forth in claim 1, wherein said L-histidinol isrecovered in the form of a crystalline salt thereof.
 7. A method as setforth in claim 6, wherein said salt is the hydrochloride.